Simultaneous Determination of Oleanolic and Ursolic Acids in Rat Plasma by HPLC-MS: Application to a Pharmacokinetic Study After Oral Administration of Different Combinations of QingGanSanJie Decoction Extracts.

A liquid chromatography-mass spectrometry method has been developed and validated for rapid simultaneous determination of the oleanolic and ursolic acid contents in rat plasma with betulinic acid as the internal standard (IS). The plasma samples were prepared by a liquid-liquid extraction procedure. Chromatographic separation was performed with a Chromasil-C18 column (250 mm × 4.6 mm, i.d. 5 µm) with methanol-water as mobile phase at 1 mL/min. The detection was accomplished under selected-ion-monitoring mode with a negative electrospray ionization interface. Linear calibration curves were obtained between the range of 0.86-421.2 and 0.94-462.0 ng/mL for oleanolic and ursolic acids, with lower limits of quantification at 0.43 and 0.47 ng/mL, respectively. The extraction recovery exceeded 70% in plasma. The intra- and interday precision values were <9.8% with the accuracy as -7.0 to 9.9% at three different QC levels in both cases. The pharmacokinetic behaviors of oral dosage of QingGanSanJie decoctions were then studied in rats following the developed approach. The t1/2 values of the oleanolic and ursolic acids after oral administration of the monarch medicine extract were significantly different (P < 0.05) from other prescription extracts containing different herb pieces with different compatibilities. Bimodal phenomena appeared in every concentration-time curve for the oleanolic and ursolic acids at 3-8 h after administration. The minister, assistant and guide medicines in the formula could prolong the metabolism of the oleanolic and ursolic acids in vivo, providing an experimental basis for the slow onset and long action of the Traditional Chinese Medicine compound.

Lipidomic profiling reveals significant alterations in lipid biochemistry in hypothyroid rat cerebellum and the therapeutic effects of Sini decoction.

Hypothyroidism is known to be closely associated with lipid metabolism. Although our previous serum and urine metabonomics studies have provided some clues about the molecular mechanism of hypothyroidism at the metabolic level, the precise mechanism underlying the pathogenesis of hypothyroidism remains elusive, especially from the aspect of lipid metabolism. In the present study, we applied an ultra high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOF-MS)-based lipidomics method to analyze the global lipid profiles of hypothyroidism in rat cerebellum. Using unsupervised analysis and multivariate statistical analysis, we separated the Sham and hypothyroid groups clearly and screened out 23 potential lipid biomarkers related to hypothyroidism that were primarily involved in sphingolipid metabolism, glycerophospholipid metabolism and ß-oxidation of fatty acid. Subsequently, we conducted computational analysis to build and simulate the lipid network of hypothyroidism, knowing that it would be useful to elucidate the pathological mechanism of hypothyroidism. Based on the selected 23 lipid biomarkers, we systematically evaluated the therapeutic effects of Sini decoction (SND) and the positive drug T4. The results showed that both SND and T4 can to some extent convert the pathological status of hypothyroidism through different pathways. Overall, this investigation illustrates that lipidomic profiling approach is powerful in giving a complementary view to the pathophysiology of hypothyroidism and offers a valuable tool for systematic study of the therapeutic effects of SND on hypothyroidism at lipid level.

Investigation of the therapeutic effectiveness of active components in Sini decoction by a comprehensive GC/LC-MS based metabolomics and network pharmacology approaches.

As a classical formula, Sini decoction (SND) has been fully proved to be clinically effective in treating doxorubicin (DOX)-induced cardiomyopathy. Current chemomics and pharmacology proved that the total alkaloids (TA), total gingerols (TG), total flavones and total saponins (TFS) are the major active ingredients of Aconitum carmichaelii, Zingiber officinale and Glycyrrhiza uralensis in SND respectively. Our animal experiments in this study demonstrated that the above active ingredients (TAGFS) were more effective than formulas formed by any one or two of the three individual components and nearly the same as SND. However, very little is known about the action mechanisms of TAGFS. Thus, this study aimed to use for the first time the combination of GC/LC-MS based metabolomics and network pharmacology for solving this problem. By metabolomics, it was found that TAGFS worked by regulating six primary pathways. Then, network pharmacology was applied to search for specific targets. 17 potential cardiovascular related targets were found through molecular docking, 11 of which were identified by references, which demonstrated the therapeutic effectiveness of TAGFS using network pharmacology. Among these targets, four targets, including phosphoinositide 3-kinase gamma, insulin receptor, ornithine aminotransferase and glucokinase, were involved in the TAGFS regulated pathways. Moreover, phosphoinositide 3-kinase gamma, insulin receptor and glucokinase were proved to be targets of active components in SND. In addition, our data indicated TA as the principal ingredient in the SND formula, whereas TG and TFS served as adjuvant ingredients. We therefore suggest that dissecting the mode of action of clinically effective formulae with the combination use of metabolomics and network pharmacology may be a good strategy.

An LC-MS/MS method for the simultaneous determination and pharmacokinetic studies of bergenin, chlorogenic acid and four flavonoids in rat plasma after oral administration of a QingGanSanJie decotion extract.

A rapid, simple and sensitive, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of bergenin, chlorogenic acid and four flavonoids in a QingGanSanJie preparation in rat plasma. Puerarin was selected as the internal standard (IS). Plasma samples were precipitated with methanol and separated with a reverse phase Agilent Poroshell 120 EC-C18 column using a gradient mobile phase of methanol-water containing 0.1% formic acid (v/v). A triple quadruple mass spectrometer was used for quantification (limit of detection 0.36-5.55 ng/mL). Intra-day and inter-day precisions were within 15% and the average extraction recoveries ranged from 85 to 115% for each analyte. The method allowed simultaneous quantification for the first time of the pharmacokinetics of bergenin, chlorogenic acid and four flavonoids after intragastric administration of a QingGanSanJie extract in Sprague-Dawley rats. It was found that bergenin and chlorogenic acid had typical extravascular administration concentration-time curves; flavonoids had a bimodal distribution improving bioavailability and extending the pharmacodynamics period.

Absorption and metabolism of three monoester-diterpenoid alkaloids in Aconitum carmichaeli after oral administration to rats by HPLC-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum carmichaelii (AC) is a well-known herbal medicine for its excellent pharmacological effects and toxicity. The monoester-diterpenoid alkaloids (MDAs), including benzoylmesaconine (BMC), benzoylaconine (BAC) and benzoylhyaconine (BHC), are the main active components in AC. It was found that the diester alkaloids could be transformed into monoester-diterpenoid alkaloids after being decocted. In Chinese pharmacopoeia, the MDAs are also used as phytochemical markers for the quality control of AC. Benzoylmesaconine, benzoylaconine and benzoylhyaconine are representatives of monoester-diterpenoid alkaloids. It was reported that the absolute bioavailability of MDAs was very low but there was toxicity often occurred in AC. Because most of DDAs are transformed into MDAs after decoction, we speculate that some other components may promote the bioavailability of MDAs but result in toxicity by enhancing their absorption. To demonstrate the dynamic changes of MDAs in vivo and reveal the causes of low bioavailability and toxicity, this study will explore the mechanisms of absorption and metabolism of 3 MDAs. MATERIALS AND METHODS: A sensitive, accurate and specific LC-MS method was developed to determine the three MDAs in rat plasma. The pharmacokinetic parameters were estimated after orally administered 3 MDAs to the Male Sprague-Dawley rats, and the metabolism stability was calculated after incubating with rat liver microsomes, finally, the absorption characteristics of the 3 MDAs were investigated using Caco-2 transwell model. RESULTS: It was found that the pharmacokinetic parameters of 3 MDAs were similar, Cmax and Tmax were very small, and t1/2 was large, which indicated 3 MDAs can be absorbed rapidly and is difficult to be metabolized or excreted. However, the low Cmax indicated that the bioavailability of 3 MDAs will be very low and their absorption may be inhibited by some transport proteins. By incubating three MDAs in rat liver microsomes, it was proved that they almost can't be metabolized in vivo. The Caco-2 transwell experiments reveal that the P-gp inhibits the absorption of MDAs. CONCLUSIONS: LC-MS combined with a direct precipitation method for the simultaneous quantification of 3 MDAs in rat plasma has been developed and validated and successfully used in pharmacokinetic study of 3 MDAs. It was proved that the three components almost can't be metabolized in vivo, and P-gp inhibits the absorption of MDAs.

Comparative normal/failing rat myocardium cell membrane chromatographic analysis system for screening specific components that counteract doxorubicin-induced heart failure from Acontium carmichaeli.

Cell membrane chromatography (CMC) derived from pathological tissues is ideal for screening specific components acting on specific diseases from complex medicines owing to the maximum simulation of in vivo drug-receptor interactions. However, there are no pathological tissue-derived CMC models that have ever been developed, as well as no visualized affinity comparison of potential active components between normal and pathological CMC columns. In this study, a novel comparative normal/failing rat myocardium CMC analysis system based on online column selection and comprehensive two-dimensional (2D) chromatography/monolithic column/time-of-flight mass spectrometry was developed for parallel comparison of the chromatographic behaviors on both normal and pathological CMC columns, as well as rapid screening of the specific therapeutic agents that counteract doxorubicin (DOX)-induced heart failure from Acontium carmichaeli (Fuzi). In total, 16 potential active alkaloid components with similar structures in Fuzi were retained on both normal and failing myocardium CMC models. Most of them had obvious decreases of affinities on failing myocardium CMC compared with normal CMC model except for four components, talatizamine (TALA), 14-acetyl-TALA, hetisine, and 14-benzoylneoline. One compound TALA with the highest affinity was isolated for further in vitro pharmacodynamic validation and target identification to validate the screen results. Voltage-dependent K(+) channel was confirmed as a binding target of TALA and 14-acetyl-TALA with high affinities. The online high throughput comparative CMC analysis method is suitable for screening specific active components from herbal medicines by increasing the specificity of screened results and can also be applied to other biological chromatography models.

Metabolic profiling provides a system understanding of hypothyroidism in rats and its application.

BACKGROUND: Hypothyroidism is a chronic condition of endocrine disorder and its precise molecular mechanism remains obscure. In spite of certain efficacy of thyroid hormone replacement therapy in treating hypothyroidism, it often results in other side effects because of its over-replacement, so it is still urgent to discover new modes of treatment for hypothyroidism. Sini decoction (SND) is a well-known formula of traditional Chinese medicine (TCM) and is considered as efficient agents against hypothyroidism. However, its holistic effect assessment and mechanistic understanding are still lacking due to its complex components. METHODOLOGY/PRINCIPAL FINDINGS: A urinary metabonomic method based on ultra performance liquid chromatography coupled to mass spectrometry was employed to explore global metabolic characters of hypothyroidism. Three typical hypothyroidism models (methimazole-, propylthiouracil- and thyroidectomy-induced hypothyroidism) were applied to elucidate the molecular mechanism of hypothyroidism. 17, 21, 19 potential biomarkers were identified with these three hypothyroidism models respectively, primarily involved in energy metabolism, amino acid metabolism, sphingolipid metabolism and purine metabolism. In order to avert the interference of drug interaction between the antithyroid drugs and SND, the thyroidectomy-induced hypothyroidism model was further used to systematically assess the therapeutic efficacy of SND on hypothyroidism. A time-dependent recovery tendency was observed in SND-treated group from the beginning of model to the end of treatment, suggesting that SND exerted a recovery effect on hypothyroidism in a time-dependent manner through partially regulating the perturbed metabolic pathways. CONCLUSIONS/SIGNIFICANCE: Our results showed that the metabonomic approach is instrumental to understand the pathophysiology of hypothyroidism and offers a valuable tool for systematically studying the therapeutic effects of SND on hypothyroidism.

Metabonomic profiles delineate the effect of traditional Chinese medicine sini decoction on myocardial infarction in rats.

BACKGROUND: In spite of great advances in target-oriented Western medicine for treating myocardial infarction (MI), it is still a leading cause of death in a worldwide epidemic. In contrast to Western medicine, Traditional Chinese medicine (TCM) uses a holistic and synergistic approach to restore the balance of Yin-Yang of body energy so the body's normal function can be restored. Sini decoction (SND) is a well-known formula of TCM which has been used to treat MI for many years. However, its holistic activity evaluation and mechanistic understanding are still lacking due to its complex components. METHODOLOGY/PRINCIPAL FINDINGS: A urinary metabonomic method based on nuclear magnetic resonance and ultra high-performance liquid chromatography coupled to mass spectrometry was developed to characterize MI-related metabolic profiles and delineate the effect of SND on MI. With Elastic Net for classification and selection of biomarkers, nineteen potential biomarkers in rat urine were screened out, primarily related to myocardial energy metabolism, including the glycolysis, citrate cycle, amino acid metabolism, purine metabolism and pyrimidine metabolism. With the altered metabolism pathways as possible drug targets, we systematically analyze the therapeutic effect of SND, which demonstrated that SND administration could provide satisfactory effect on MI through partially regulating the perturbed myocardial energy metabolism. CONCLUSIONS/SIGNIFICANCE: Our results showed that metabonomic approach offers a useful tool to identify MI-related biomarkers and provides a new methodological cue for systematically dissecting the underlying efficacies and mechanisms of TCM in treating MI.

Detection and identification of diterpenoid alkaloids, isoflavonoids and saponins in Qifu decoction and rat plasma by liquid chromatography-time-of-flight mass spectrometry.

A liquid chromatography-time-of-flight mass spectrometric (LC-TOFMS) method has been developed for analysis of components in Qifu decoction (QFD), a traditional Chinese medical formula consisting of Radix Astragali and Acontium carmichaeli, and in rat plasma after oral administration. Based on accurate mass measurements within 3 ppm error for each molecular ion and subsequent fragment ions of TOFMS, as well as matching of empirical molecular formulae with those of published components in the in-house chemical library, a total of 44 major components including 21 diterpenoid alkaloids, 12 flavonoids and 11 saponins were identified in QFD. After oral administration of QFD, 22 components in rat plasma were detected and identified by comparing and contrasting the constituents measured in QFD with those in the plasma samples. The results provided valuable chemical information for further pharmacology and active mechanism research on QFD. LC-TOFMS was also applied for the comparison of relative peak area of major active components between QFD and the single herb extracts. The concentration ratios of major saponins detected in the crude herb Radix Astragali were found to be different from those in QFD. The experimental data indicated that the decocting process could result in differences in the amounts of active components.

Hydrophilic interaction and reversed-phase ultraperformance liquid chromatography TOF-MS for serum metabonomic analysis of myocardial infarction in rats and its applications.

An ultra performance liquid chromatography coupled to mass spectrometry-based metabonomic approach, which utilizes both reversed-performance (RP) chromatography and hydrophilic interaction chromatography (HILIC) separations, has been developed to characterize the global serum metabolic profile associated with myocardial infarction (MI). The HILIC was found necessary for a comprehensive serum metabonomic profiling, providing complementary information to RP chromatography. By combining with partial least squares discriminant analysis, 21 potential biomarkers in rat serum were identified. To further elucidate the pathophysiology of MI, related metabolic pathways have been studied. It was found that MI was closely related to disturbed sphingolipid metabolism, phospholipid catabolism, fatty acid transportation and metabolism, tryptophan metabolism, branched-chain amino acids metabolism, phenylalanine metabolism, and arginine and proline metabolism. With the presented metabonomic method, we systematically analyzed the therapeutic effects of Traditional Chinese Medicine Sini decoction (SND). The results demonstrated that SND administration could provide satisfactory effects on MI through partially regulating the perturbed metabolic pathways.

Potential biomarkers in mouse myocardium of doxorubicin-induced cardiomyopathy: a metabonomic method and its application.

BACKGROUND: Doxorubicin (DOX) is one of the most potent antitumor agents available; however, its clinical use is limited because of the risk of severe cardiotoxicity. Though numerous studies have ascribed DOX cardiomyopathy to specific cellular pathways, the precise mechanism remains obscure. Sini decoction (SND) is a well-known formula of Traditional Chinese Medicine (TCM) and is considered as efficient agents against DOX-induced cardiomyopathy. However, its action mechanisms are not well known due to its complex components. METHODOLOGY/PRINCIPAL FINDINGS: A tissue-targeted metabonomic method using gas chromatography-mass spectrometry was developed to characterize the metabolic profile of DOX-induced cardiomyopathy in mice. With Elastic Net for classification and selection of biomarkers, twenty-four metabolites corresponding to DOX-induced cardiomyopathy were screened out, primarily involving glycolysis, lipid metabolism, citrate cycle, and some amino acids metabolism. With these altered metabolic pathways as possible drug targets, we systematically analyzed the protective effect of TCM SND, which showed that SND administration could provide satisfactory effect on DOX-induced cardiomyopathy through partially regulating the perturbed metabolic pathways. CONCLUSIONS/SIGNIFICANCE: The results of the present study not only gave rise to a systematic view of the development of DOX-induced cardiomyopathy but also provided the theoretical basis to prevent or modify expected damage.

Identification of multiple components in Guanxinning injection using hydrophilic interaction liquid chromatography/time-of-flight mass spectrometry and reversed-phase liquid chromatography/time-of-flight mass spectrometry.

An approach for the identification of multiple components in traditional Chinese medicine injections (TCMIs) using a combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) coupled with time-of-flight mass spectrometry (TOFMS) was developed for the quality control of Guanxinning injection (GXNI), a widely used TCMI, composed of Salvia miltiorrhiza and Ligusticum Chuanxiong. A total of 50 compounds from five compound classes, including saccharides, amino acids, organic acids, phenolic acids and phthalides, were identified or tentatively characterized on the basis of accurate mass measurements and subsequent TOFMS product ions. Six groups of isomers of phenolic acids and saccharides were tentatively distinguished. It was observed that the ESI-TOFMS fragmentation behavior of phthalides was different in negative and positive ion mode, and the fragmentation pathways were tentatively elucidated using structurally-relevant product ions. Several highly polar constituents were characterized for the first time from GXNI by HILIC/TOFMS. In addition, all the constituents identified from GXNI were further assigned in the two individual crude drugs. The integrated strategy has provided a powerful approach for the separation and identification of the multiple components in GXNI, and it has also assisted in the establishment of methods for the comprehensive safety and quality evaluation of TCMIs.

Screening and analysis of aconitum alkaloids and their metabolites in rat urine after oral administration of aconite roots extract using LC-TOFMS-based metabolomics.

Aconite roots are popularly used in herbal medicines in China. Many cases of accidental and intentional intoxication with this plant have been reported; some of these are fatal because the toxicity of aconitum is very high. It is thus important to detect and identify aconitum alkaloids in biofluids. In this work, an improved method employing LC-TOFMS with multivariate data analysis was developed for screening and analysis of major aconitum alkaloids and their metabolites in rat urine following oral administration of aconite roots extract. Thirty-four signals highlighted by multivariate statistical analyses including 24 parent components and 10 metabolites were screened out and further identified by adjustment of the fragmentor voltage to produce structure-relevant fragment ions. It is helpful for studying aconite roots in toxicology, pharmacology and forensic medicine. This work also confirmed that the metabolomic approach provides effective tools for screening multiple absorbed and metabolic components of Chinese herbal medicines in vivo.